1. Technical Field
The present invention generally refers to magnetic nanoparticle sensors and to molecular recognition procedures, suitable for applications in several different fields.
As such, the biomolecular recognition is the interaction between biomolecules, which have a mutual affinity or present some sort of complementarity. Examples of such interactions are the DNA-DNA hybridization, the antibody-antigen recognition and the ligand-receptor bond [H. A. Ferreira et al., IEEE Transactions on Magnetics 41, 4140 (2005)].
2. Description of the Related Art
The biomolecular recognition is, actually, omnipresent in life, as it constitutes the way the cell mechanisms work. Evident examples are the genetic code replication, the enzyme generation, protein glycids and nucleic acids fabrication and modification, the intracellular and extracellular transport, the cellular metabolism, to mention some of the most common biological processes. On the other hand, more familiar uses of the biomolecular recognition comprise pregnancy tests, blood group verification, genetic screening and site-directed cancer treatments, currently under development. It is clear that the biomolecular recognition detection is more and more important in fields such as healthcare, pharmaceutical industry, environmental analysis, and in general in biotechnological applications.
In general, the detection results in the usage of a known biomolecule which probes a test sample, looking for a specific target analyte. A common approach for detecting biological molecules is to attach a marker to the target molecule, which produces an externally observable signal. Traditionally, this is implemented by using a molecular recognition between the target molecule and a specific receptor (for example an antibody) labelled by the marker. The marker can be a radioisotope, an enzyme or a fluorescent molecule, as in case of the LIF (Light Induced Fluorescence) techniques. Recently, as markers for the bio-detection, magnetic microparticles and nanoparticles have also been used, due to their advantages in respect to the other markers. The magnetic properties of the nanoparticles are stable over the time, since the magnetism is neither affected by the chemistry of the reagents, nor subject to photobleaching (a problem related to the use of fluorescent markers). Moreover, in biological applications, generally, there is no significant magnetic background signal, and the magnetic fields are not screened by reagents or aqueous biomaterials. Moreover, magnetism can be used to remotely handle the magnetic particles. It is to be highlighted that the sizes of the magnetic particles shall be as reduced as possible for introducing as little perturbation as possible in relation to the affinity between the probe molecules and the target molecules. In any case, it is necessary to find a compromise between the latter aspect and the need of a magnetic moment, dimensioned such that it can be detected by a specific sensor with a proper signal-to-noise ratio. Until now, several research groups have used particles with a diameter from a few microns to 16 nm.
In the last years, a lot of very sensible magnetic fields detecting devices have been developed, such as giant magnetoresistance (GMR) [M. N. Baibich et al., Phys. Rev. Lett. 61 (21), 2472-2475 (1998)] and spin valve magnetic sensors [B. Dieny et al., J. Appl. Phys. 69(8), 4774 (1991); P. P. Freitas et al., Sens Actuat A Phys, 81 (1-3), 2 (2000)] which allow extremely weak magnetic fields to be measured, such as those generated by a single magnetic microparticle. Beside the GMR sensors, detection of single magnetic particles has been demonstrated with miniaturized silicon Hall sensors [P. A. Besse et al., Appl. Phys. Lett. 80 (22), 4199 (2002)] and planar Hall effect sensors, based on thin Permalloy films (FeNi alloy) [L. Ejsing et al., Appl. Phys. Lett. 84 (23), 4729 (2004)]. More recently, tunnel magnetoresistance (TMR) sensors have been applied to the molecular recognition, based on magnetic tunnel junctions (MTJ) [W. Shen et al., Appl. Phys. Lett. 2008, 103, 07A306], due to their superior sensibility and stability in relation to temperature. The use of anisotropic magnetoresistance in ring sensors, as sensitive particle detectors, has been suggested by Miller et al. [M. M. Miller et al., Appl. Phys. Lett. 81, 2211 (2002)] and, more recently, such an approach has been extended by L. Llandro et al. [L. Llandro et al., Appl. Phys. Lett. 91, 203904 (2007)] to multi-layer ring sensors (pseudo-spin valve) based on the giant magnetoresistance (GMR) effect. In the first case, the ring sensor was made of NiFe and it was designed to detect the radial component of the field created by a single microsphere (diameter 4.3 microns), magnetized by an alternating magnetic field. In the second case, the detection of a superparamagnetic microsphere with a diameter of 4 microns was reported, by using a pseudo-spin valve ring sensor. In absence of the particle, the magnetization of the sensor free layer is modified by a periodical external magnetic field, so that, every time an anti-parallel alignment configuration of the magnetic layers is established, a GMR peak is generated. Once a magnetic particle is placed on the sensor, the particle screens the external magnetic field, and causes a change of the external fields needed for the reversal of the free layer. In order to compare several sensors for the biomolecular recognition, the following parameters have to be taken into account:                1) Biological sensitivity—it corresponds to the ability to detect low concentrations of the target analyte in biological samples, and it is normally given in terms of limit of detection (LOD); a low LOD corresponds to a high biological sensitivity;        2) Dynamic range—such a parameter indicates the analyte concentration range, which can be detected by the sensor, operating on a linear regime in a given biological assay.        
In some cases, the biological sensitivity is not a problem, as amplification processes, which enable an increase in the analyte concentration, such as the polymerase chain reaction (PCR), for the DNA in genomics, can be applied. In other applications (such as proteomics), however, there are no suitable methods for amplification, therefore a high biological sensitivity is required.
The dynamic range is a feature, which is often in competition with the biological sensitivity. Sensors, designed for detecting individual particles, are not able to count large numbers of molecules, so their application in analyte concentration measurements becomes troublesome.
Moreover, the paper of P. Vavassori et al., Appl. Phys. Lett 91, 093114 (2007), regarding square rings of Permalloy applied in the magnetic storage field, is useful to fully understand the teachings of the present invention.